构建TRAF6基因3’非编码区荧光素酶报告质粒验证及与潜在靶基因TRAF6的靶向关系
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摘要
背景:前期研究发现miR-146-5p在染氟成骨细胞凋亡时表达上调。目的:构建肿瘤坏死因子受体相关因子6(TNF receptor associated factor 6,TRAF6)基因3’非编码区(3'UTR)荧光素酶报告质粒,利用双荧光素酶报告基因验证miR-146-5p与其潜在靶基因TRAF6的靶向关系。方法:采用生物信息学方法预测mi R-146-5p与TRAF6基因的结合位点。采用PCR技术扩增TRAF6基因3'UTR片段,并克隆至p Yr-MirTarget载体,构建野生型重组双荧光素酶报告质粒。实验分6组:①6a-5p-核苷类似物+TRAF6野生型3'-UTR共转组;②无核苷类似物对照+TRAF6野生型3'UTR共转组(对照组);③miR-146a-5p-抑制剂+TRAF6野生型;④TRAF6野生型3'-UTR转染组;⑤p Yr-MirTarget空质粒转染组;⑥正常细胞组。分别将重组荧光素酶报告质粒与miR-146-5p和核苷类似物共转染Saos-2,同时设置无核苷类似物对照、miR-146-5p抑制剂阴性对照和空载p Yr-MirTarget-W3'UTR、TRAF6-W 3'UTR及正常对照。检测6组细胞中荧光素酶活性。将核苷类似物和miR-146-5pinhibitor以及无核苷类似物对照分别转染人成骨细胞Saos-2,裂解细胞提取蛋白后采用免疫印迹检测TRAF6蛋白表达水平。结果与结论:①共转染miR-146-5p mimics的Saos-2细胞中荧光素酶活性为10.103 0±0.558 5、对照组(无核苷类似物-TRAF63'UTR)荧光素酶活性为13.1400±0.7204、抑制剂(inhibitor)组荧光素酶活性为13.707 1±0.434 8、野生型TRAF6 3'UTR质粒的Saos-2细胞中荧光素酶活性为13.202 1±0.456 5。仅转染空质粒的细胞荧光素梅活性为14.706 2±0.441 6,Saos-2细胞中荧光素酶活性本底值为1.126 4±0.126 2。共转染miR-146-5p mimics组明显降低(F=715.789,P <0.000 1),其他3组间与对照组比较,差异无统计学意义(P> 0.05);②免疫印迹结果显示,与对照组比较,细胞转染miR-146-5p mimics后TRAF6蛋白表达水平明显下调;③结果说明,TRAF6与mi R-146-5p存在靶向关系。
BACKGROUND: Preliminary study has found that miR-146-5 p is up-regulated in osteobIasts induced by sodium fIuoride.OBJECTIVE: To verify the targeting relationship between miR-146-5 p and its potential target gene TNF receptor associated factor 6(TRAF6)using a dual luciferase reporter gene through constructing a luciferase reporter plasmid for the 3' non-coding region(3'UTR) of TRAF6 gene.METHODS: Bioinformatics methods were used to predict the binding sites of miR-146-5 p and TRAF6 genes. The 3'UTR fragment of TRAF6 gene was amplified by PCR and cloned into pYr-MirTarget vector to construct a wild type recombinant dual-luciferase reporter plasmid. There were six groups:(1) 6a-5p-nucleoside analogs + TRAF6-W 3'UTR co-transfection group;(2) non-nucleoside analogs mimics + TRAF6-W3'UTR co-transfection group(control group);(3) miR-146 a-5p inhibitor + TRAF6-W group;(4) TRAF6-W 3'UTR co-transfection group;(5)empty pYr-MirTarget transfection group;(6) normal cell group. The co-transfection of recombinant luciferase reporter plasmid and miR-146-5 p or nucleoside analogs(mimics) with Saos-2 was made, respectively. At the same time, nucleoside analogs mimics(control group),miR-146-5p inhibitor(negative control group) and empty pYr-MirTarget-W 3'UTR and TRAF6-W 3'UTR were added simultaneously to detect luciferase activity in six groups of cells. Mimics, miR-146-5p inhibitor and nucleoside analogs mimics were transfected to the human osteoblast Saos-2 respectively. The protein expression level was detected by western blot assay after lysing cells to extract protein.RESULTS AND CONCLUSION:(1) Luciferase activity in Saos-2 cells co-transfected with miR-146-5p mimics was 10.103 0±0.558 5; the control group's(NC mimics-TRAF6 3'UTR) luciferase activity was 13.140 0±0.720 4; the inhibitor group's luciferase activity was 13.707 1±0.434 8; and luciferase activity in Saos-2 cells of the wild-type TRAF6 3'UTR plasmid was 13.202 1±0.456 5. The luciferase activity of the cells transfected with the empty plasmid alone was 14.706 2±0.441 6. The original luciferase activity in Saos-2 cells was 1.126 4±0.126 2. The number of miR-146-5 p mimics co-transfected significantly decreased(F=715.789, P < 0.000 1). The other three groups were compared with the control group, showing no significant difference(P > 0.05).(2) Western blot assay showed that compared with the control group, the expression level of TRAF6 protein was significantly down-regulated after transfection of miR-146-5 p mimics.(3) To conclude, there is a targeted relationship between TRAF6 and mi R-146-5 p.
BACKGROUND: Preliminary study has found that miR-146-5 p is up-regulated in osteobIasts induced by sodium fIuoride.OBJECTIVE: To verify the targeting relationship between miR-146-5 p and its potential target gene TNF receptor associated factor 6(TRAF6)using a dual luciferase reporter gene through constructing a luciferase reporter plasmid for the 3' non-coding region(3'UTR) of TRAF6 gene.METHODS: Bioinformatics methods were used to predict the binding sites of miR-146-5 p and TRAF6 genes. The 3'UTR fragment of TRAF6 gene was amplified by PCR and cloned into pYr-MirTarget vector to construct a wild type recombinant dual-luciferase reporter plasmid. There were six groups:(1) 6a-5p-nucleoside analogs + TRAF6-W 3'UTR co-transfection group;(2) non-nucleoside analogs mimics + TRAF6-W3'UTR co-transfection group(control group);(3) miR-146 a-5p inhibitor + TRAF6-W group;(4) TRAF6-W 3'UTR co-transfection group;(5)empty pYr-MirTarget transfection group;(6) normal cell group. The co-transfection of recombinant luciferase reporter plasmid and miR-146-5 p or nucleoside analogs(mimics) with Saos-2 was made, respectively. At the same time, nucleoside analogs mimics(control group),miR-146-5p inhibitor(negative control group) and empty pYr-MirTarget-W 3'UTR and TRAF6-W 3'UTR were added simultaneously to detect luciferase activity in six groups of cells. Mimics, miR-146-5p inhibitor and nucleoside analogs mimics were transfected to the human osteoblast Saos-2 respectively. The protein expression level was detected by western blot assay after lysing cells to extract protein.RESULTS AND CONCLUSION:(1) Luciferase activity in Saos-2 cells co-transfected with miR-146-5p mimics was 10.103 0±0.558 5; the control group's(NC mimics-TRAF6 3'UTR) luciferase activity was 13.140 0±0.720 4; the inhibitor group's luciferase activity was 13.707 1±0.434 8; and luciferase activity in Saos-2 cells of the wild-type TRAF6 3'UTR plasmid was 13.202 1±0.456 5. The luciferase activity of the cells transfected with the empty plasmid alone was 14.706 2±0.441 6. The original luciferase activity in Saos-2 cells was 1.126 4±0.126 2. The number of miR-146-5 p mimics co-transfected significantly decreased(F=715.789, P < 0.000 1). The other three groups were compared with the control group, showing no significant difference(P > 0.05).(2) Western blot assay showed that compared with the control group, the expression level of TRAF6 protein was significantly down-regulated after transfection of miR-146-5 p mimics.(3) To conclude, there is a targeted relationship between TRAF6 and mi R-146-5 p.
引文
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[15]Li J,Zhao L,Zhao X,et al.Foxo1 Attenuates NaF-Induced Apoptosis of LS8 Cells through the JNK and Mitochondrial Pathways.Biol Trace Elem Res.2018;181(1):104-111.
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[17]陈荣,于燕妮,徐淋,等.mTOR自噬通路在染氟大鼠软骨关节损伤中的作用[J].中国地方病防治杂志2017,32(1):18-19.
[18]Wang Y,Li Y.miR-146 promotes HBV replication and expression by targeting ZEB2.Biomed Pharmacother.2018;99:576-582.
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[21]Li P,Sun N,Zeng J,et al.Differential expression of miR-672-5p and miR-146a-5p in osteoblasts in rats after steroid intervention.Gene.2016;591(1):69-73.
[22]Waki T,Lee SY,Niikura T,et al.Profiling microRNAexpression in fracture nonunions:potential role of microRNAs in nonunion formation studied in a rat model.Bone Joint J.2015;97-B(8),1144-1151.
[23]Xi Y,Jiang T,Wang W,et al.Long non-coding HCG18promotes intervertebral disc degeneration by sponging miR-146a-5p and regulating TRAF6 expression.Sci Rep.2017;7(1):13234
[24]Xu B,Huang Y,Niu X,et al.Hsa-miR-146a-5p modulates androgen-independent prostate cancer cells apoptosis by targeting ROCK1.Prostate.2015;75(16):1896-903.
[25]Pan Q,Liu H,Zheng C,et al.Microvesicles Derived from Inflammation-Challenged Endothelial Cells Modulate Vascular Smooth Muscle Cell Functions.Front Physiol.2017;7:692.
[26]Li H,Xie S,Liu M,et al.The clinical significance of downregulation of mir-1243p,mir-146a-5p,mir-155-5p and mir-335-5p in gastric cancer tumorigenesis.Int J Oncol.2014;45(1),197-208.
[27]Lu Y,Cao L,Jiang,B.C,et al.,MicroRNA-146a-5p attenuates neuropathic pain via suppressing TRAF6 signaling in the spinal cord.Brain BehavImmun.2015;49,119-129.
[28]王珊,李晓霞,周杰,等.Nfic基因3’UTR双荧光素酶报告质粒的构建及其与miR-20a靶向关系的验证[J].天津医药,2016,9(44):1065-1068
[29]Lee JY,Kim S,Hwang DW,et al.Development of a dual-luciferase reporter system for in vivo visualization of MicroRNA biogenesis and posttranscriptional regulation.JNucl Med.2008;49(2):285-94.
[30]Wu GQ,Wang X,Zhou HY,et al.Evidence for transcriptional interference in a dual-luciferase reporter system.Sci Rep.2015;5:17675.
[2]Toh WS,Lai RC,Zhang B,etal.MSC exosome works through a protein-based mechanism of action.BiochemSoc Trans.2018;46(4):843-853
[3]Zhong X,Zhang D,Xiong M,et al.Noncoding RNA for Cancer Gene Therapy.Recent Results Cancer Res.2016;209:51-60.
[4]Yao S.MicroRNA biogenesis and their functions in regulating stem cell potency and differentiation.BiolProced Online.2016;18:1-10.
[5]邓强,张亚楼,周杨俊杰,等.氟化钠诱导人成骨细胞凋亡相关microRNA差异表达及分析[J].中国组织工程研究,2018,22(8):1149-1154.
[6]Min SK,Jung SY,Kang HK,et al.Functional diversity of miR-146a-5p and TRAF6 in normal and oral cancer cells.Int JOncol.2017;51(5):1541-1552.
[7]Peng S,Gao D,Gao C,et al.MicroRNAs regulate signaling pathways in osteogenic differentiation of mesenchymal stem cells.Mol Med Rep.2016;14(1):623-9.
[8]Wang W,Wang X,Chen L,et al.The microRNA miR-124suppresses seizure activity and regulates CREB1 activity.Expert Rev Mol Med.2016;18:e4.
[9]Nicolas FE.Experimental validation of microRNA targets using a luciferase reporter system.Methods Mol Biol.2011;732:139-52.
[10]Huntley RP,Kramarz B,Sawford T,et al.Expanding the horizons of microRNA bioinformatics.RNA.2018,24(8):1005-1017.
[11]马国福.氟骨症临床与影像学诊断[J].影像研究与医学应用,2017,1(12):55-56.
[12]史睿亭,李维维,徐泽华.地方性氟骨症治疗的研究进展[J].生物技术世界,2015,(5):81.
[13]Lima VV,de Almeida Carrer FC,Gabriel M,et al.Knowledge of primary care professionals about fluoride topics.Minerva Stomatol.2016;67(5):196-201.
[14]Liu L1,Zhang Y,GuH,et al.Fluorosis induces endoplasmic reticulum stress and apoptosis in osteoblasts in vivo[J].Biol Trace Elem Res.2015;164(1):64-71.
[15]Li J,Zhao L,Zhao X,et al.Foxo1 Attenuates NaF-Induced Apoptosis of LS8 Cells through the JNK and Mitochondrial Pathways.Biol Trace Elem Res.2018;181(1):104-111.
[16]Suzuki M,Bandoski C,Bartlett JD.Fluoride induces oxidative damage and SIRT1/autophagy through ROS-mediated JNKsignaling.Free Radic Biol Med.2015,89:369-378.
[17]陈荣,于燕妮,徐淋,等.mTOR自噬通路在染氟大鼠软骨关节损伤中的作用[J].中国地方病防治杂志2017,32(1):18-19.
[18]Wang Y,Li Y.miR-146 promotes HBV replication and expression by targeting ZEB2.Biomed Pharmacother.2018;99:576-582.
[19]Testa U,Pelosi E,Castelli G,et al.miR-146 and miR-155:Two Key Modulators of Immune Response and Tumor Development.Noncoding RNA.2017;3(3).pii:E22.
[20]Wang H,Zhang Y,Wu X,et al.Regulation of Human Natural Killer Cell IFN-γProduction by MicroRNA-146a via Targeting the NF-κB Signaling Pathway.Front Immunol.2018;9:293.
[21]Li P,Sun N,Zeng J,et al.Differential expression of miR-672-5p and miR-146a-5p in osteoblasts in rats after steroid intervention.Gene.2016;591(1):69-73.
[22]Waki T,Lee SY,Niikura T,et al.Profiling microRNAexpression in fracture nonunions:potential role of microRNAs in nonunion formation studied in a rat model.Bone Joint J.2015;97-B(8),1144-1151.
[23]Xi Y,Jiang T,Wang W,et al.Long non-coding HCG18promotes intervertebral disc degeneration by sponging miR-146a-5p and regulating TRAF6 expression.Sci Rep.2017;7(1):13234
[24]Xu B,Huang Y,Niu X,et al.Hsa-miR-146a-5p modulates androgen-independent prostate cancer cells apoptosis by targeting ROCK1.Prostate.2015;75(16):1896-903.
[25]Pan Q,Liu H,Zheng C,et al.Microvesicles Derived from Inflammation-Challenged Endothelial Cells Modulate Vascular Smooth Muscle Cell Functions.Front Physiol.2017;7:692.
[26]Li H,Xie S,Liu M,et al.The clinical significance of downregulation of mir-1243p,mir-146a-5p,mir-155-5p and mir-335-5p in gastric cancer tumorigenesis.Int J Oncol.2014;45(1),197-208.
[27]Lu Y,Cao L,Jiang,B.C,et al.,MicroRNA-146a-5p attenuates neuropathic pain via suppressing TRAF6 signaling in the spinal cord.Brain BehavImmun.2015;49,119-129.
[28]王珊,李晓霞,周杰,等.Nfic基因3’UTR双荧光素酶报告质粒的构建及其与miR-20a靶向关系的验证[J].天津医药,2016,9(44):1065-1068
[29]Lee JY,Kim S,Hwang DW,et al.Development of a dual-luciferase reporter system for in vivo visualization of MicroRNA biogenesis and posttranscriptional regulation.JNucl Med.2008;49(2):285-94.
[30]Wu GQ,Wang X,Zhou HY,et al.Evidence for transcriptional interference in a dual-luciferase reporter system.Sci Rep.2015;5:17675.